Title: single-laboratory validation of a UPLC-MS/MS based method for the detection and quantification of DHM in mouse serum and lung tissues An administrative supplement request to R01CA193278 (05/2015 ? 04/2020) ABSTRACT of the supplement DHM (dihydromethysticin) was identified as the key active ingredient in kava that completely blocks NNK-induced lung tumorigenesis in A/J mice at a human relevant dose while DHK (dihydrokavain), a structurally similar compound in kava, is completely ineffective. This clear structure-activity relationship demonstrates the importance of the methylenedioxy functional group in DHM that is absent in DHK and provides outstanding opportunity to elucidate the underlying mechanism of action. The parent R01 proposes to determine the structural determinants of DHM via the synthesis of three probe compounds, the confirmation of their similar bioavailability, and the evaluation of their in vivo chemopreventive activity in Aim 1. A specific, precise, accurate and sensitive quantitative method is needed to quantify DHM and its analogs in both mouse serum and lung tissues so that we can determine whether bioavailability is one potential mechanism responsible for the distinct efficacy differences in vivo. We therefore have developed an ultraperformance liquid chromatography high-resolution tandem mass spectrometry (UPLC-MS/MS) method for DHM detection and quantification with [2H2]-DHM (synthesized in- house) as the internal standard. The integration of the high-resolution mass spectrometry, the MS/MS fingerprints and the use of the isotope-labeled internal standard offered the unambiguous detection and accurate quantification of DHM. The method has also demonstrated a high sensitivity, an excellent recovery from both serum and lung tissues, and a wide linear range of detection and quantification. Such a method is expected to have a wide range of applications besides its application for the parent R01. First of all, it can be used to quantify DHM in different kava products for quality control and quality assurance. Secondly, it can be employed to perform pharmacokinetic studies of DHM or kava in animal models or in humans. In addition, given that DHM is only reported to be detected in kava, DHM detection in human body fluids can be used to determine kava exposure, particularly in human trials to confirm kava/DHM compliance as in the pilot human trial we have recently completed (not part of the parent R01). Lastly, as the chempreventive ingredient against lung tumorigenesis, the levels of DHM in different individuals may be predictive of the extent of lung cancer risk reduction in future clinical development. We therefore propose to perform a single-lab validation of this method, focusing on mouse serum and lung tissues as the matrixes, which are supported and needed by the parent R01. In the future, we will extend the method validation in human urine and plasma samples collected from the pilot human trial and different kava products (such as roots and capsules).